Macadamia (Macadamia integrifolia, M. tetraphylla and hybrids) is an Australian native nut crop and has a significant economic value in the food industries worldwide. Long juvenility along with ...traditional breeding strategies impede quick genetic improvement of this crop. The existing cultivars constitute only second to fourth generation of the wild germplasm in the rainforest. The utilisation of molecular markers for genomic selection and genome-wide association studies may accelerate genetic gains. Identification of a robust, reproducible, and cost-effective marker system is instrumental in increasing the efficiency of genomic studies. This study is the first to report the potential of two ultra-high-throughput diversity array technology (DArT) markers (silicoDArT and SNP) in macadamia. Both markers were used to identify the genetic diversity and population structure in 80 macadamia cultivars. Parentage analysis of 25 scions in a rootstock trial was conducted to confirm plant identity where recorded identities did not corroborate with phenotypic field observations. A total of 22,280 silicoDArT and 7,332 SNP markers were reported, of which 11,526 silicoDArT and 3,956 SNP markers were used for analyses after screening with quality control parameters including >95% call rate, >95% reproducibility, and >0.05 one ratio. The average polymorphic information content (PIC) values of silicoDArT and SNP markers were 0.29 and 0.21, respectively. Genetic variance among the cultivars ranged from 0.003 to 0.738 in silicoDArT and 0.004 to 0.412 in SNP markers. Four distinct population groups were identified from SNP data analysis. Most of the accessions used in this study were descended from two or more populations. Cluster analysis clearly separated genotypes of distinct origins, such as the Hawaii Agricultural Experiment Station and Hidden Valley Plantation accessions. Two wild accessions of Macadamia jansenii and M. ternifolia were found to be distantly related to the cultivars. Wild germplasm individuals and their hybrids with cv. '660' formed separate clusters, suggesting that crossing between wild and cultivated genepools can extend genetic diversity. DArTseq-based SNP markers were successfully utilized to confirm the genetic identity of 25 scions in a rootstock trial. Our study suggests that DArT platforms are a robust system for the facilitation of genomic studies with regard to macadamia.
SUMMARY
Recent advances in the sequencing and assembly of plant genomes have allowed the generation of genomes with increasing contiguity and sequence accuracy. Chromosome level genome assemblies ...using sequence contigs generated from long read sequencing have involved the use of proximity analysis (Hi‐C) or traditional genetic maps to guide the placement of sequence contigs within chromosomes. The development of highly accurate long reads by repeated sequencing of circularized DNA (HiFi; PacBio) has greatly increased the size of contigs. We now report the use of HiFiasm to assemble the genome of Macadamia jansenii, a genome that has been used as a model to test sequencing and assembly. This achieved almost complete chromosome level assembly from the sequence data alone without the need for higher level chromosome map information. Eight of the 14 chromosomes were represented by a single large contig (six with telomere repeats at both ends) and the other six assembled from two to four main contigs. The small number of chromosome breaks appears to be the result of highly repetitive regions including ribosomal genes that cannot be assembled by these approaches. De novo assembly of near complete chromosome level plant genomes now appears possible using these sequencing and assembly tools. Further targeted strategies might allow these remaining gaps to be closed.
Significance Statement
De novo assembly of a near compete chromosome level plant genome is now possible using long read sequencing and assembly tools.
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•The background of palmitoleic acid and macadamia nuts.•The biological functions of palmitoleic acid relationship between palmitoleic acid and health.•A possible de novo biosynthetic ...pathway of palmitoleic acid in macadamia is proposed.•A possible solution to find candidate genes responsible for the accumulation of palmitoleic acid in macadamia using GWAS is proposed.
Palmitoleic acid is an unusual omega-7 monounsaturated fatty acid that occurs naturally in high levels in macadamia plants. Due to its wide public acceptance and being a commonly occurring food, macadamia nuts may serve as an important dietary agent for the delivery of palmitoleic acid to the human body. This review focuses on discussing the biological functions of palmitoleic acid, the association of palmitoleic acid with various diseases, as well as the anticipated biosynthetic pathway of palmitoleic acid in macadamia plants. In addition, given the importance to regulate palmitoleic acid contents in macadamia to achieve improved nutrition and commercial value, this review also proposes GWAS as an effective strategy to identify the candidate genes responsible for the accumulation of palmitoleic acid in macadamia nuts.
Improving yield prediction and selection efficiency is critical for tree breeding. This is vital for macadamia trees with the time from crossing to production of new cultivars being almost a quarter ...of a century. Genomic selection (GS) is a useful tool in plant breeding, particularly with perennial trees, contributing to an increased rate of genetic gain and reducing the length of the breeding cycle. We investigated the potential of using GS methods to increase genetic gain and accelerate selection efficiency in the Australian macadamia breeding program with comparison to traditional breeding methods. This study evaluated the prediction accuracy of GS in a macadamia breeding population of 295 full-sib progeny from 32 families (29 parents, reciprocals combined), along with a subset of parents. Historical yield data for tree ages 5 to 8 years were used in the study, along with a set of 4113 SNP markers. The traits of focus were average nut yield from tree ages 5 to 8 years and yield stability, measured as the standard deviation of yield over these 4 years. GBLUP GS models were used to obtain genomic estimated breeding values for each genotype, with a five-fold cross-validation method and two techniques: prediction across related populations and prediction across unrelated populations.
Narrow-sense heritability of yield and yield stability was low (h
= 0.30 and 0.04, respectively). Prediction accuracy for yield was 0.57 for predictions across related populations and 0.14 when predicted across unrelated populations. Accuracy of prediction of yield stability was high (r = 0.79) for predictions across related populations. Predicted genetic gain of yield using GS in related populations was 474 g/year, more than double that of traditional breeding methods (226 g/year), due to the halving of generation length from 8 to 4 years.
The results of this study indicate that the incorporation of GS for yield into the Australian macadamia breeding program may accelerate genetic gain due to reduction in generation length, though the cost of genotyping appears to be a constraint at present.
Husk spot, a fungal disease of macadamia pericarps (
), induces premature abscission in several major commercial cultivars. Breeding for resistance to husk spot is a priority of the Australian ...macadamia industry. Due to the large tree size of macadamia and high numbers of progeny in breeding populations, inoculating for resistance screening is laborious and time consuming. Previously utilized methods included direct applications of
suspensions and the hanging of bags of diseased husks above developing fruit in tree canopies. In this study, both methods were modified to allow for efficient application in large-scale breeding populations, and their efficacy was evaluated. Two quantities of diseased husk per bag, 'large' (75 g) and 'small' (30 g), and two concentrations of sprayed
suspensions, 'stock' (5 × 10
propagules/ml) and 'dilute' (2.5 × 10
propagules/ml), were tested across two fruiting seasons. Treatments were compared against a control (sterile water) in commercial cultivars A38 and A4. Husk spot incidence and severity produced by small bags were significantly affected by season. A significant season effect was less common for other treatments. All four treatments infected over 50% of target fruit in each season, but the highest husk spot incidence across both seasons (≥85%) was produced from large bags. Overall, the large bags were the most reliable method for infection of target fruit. Results also demonstrate the importance of considering the effect of season when selecting husk spot inoculation methods.
Breeding for new macadamia cultivars with high nut yield is expensive in terms of time, labour and cost. Most trees set nuts after four to five years, and candidate varieties for breeding are ...evaluated for at least eight years for various traits. Genome-wide association studies (GWAS) are promising methods to reduce evaluation and selection cycles by identifying genetic markers linked with key traits, potentially enabling early selection through marker-assisted selection. This study used 295 progeny from 32 full-sib families and 29 parents (18 phenotyped) which were planted across four sites, with each tree genotyped for 4113 SNPs. ASReml-R was used to perform association analyses with linear mixed models including a genomic relationship matrix to account for population structure. Traits investigated were: nut weight (NW), kernel weight (KW), kernel recovery (KR), percentage of whole kernels (WK), tree trunk circumference (TC), percentage of racemes that survived from flowering through to nut set, and number of nuts per raceme.
Seven SNPs were significantly associated with NW (at a genome-wide false discovery rate of < 0.05), and four with WK. Multiple regression, as well as mapping of markers to genome assembly scaffolds suggested that some SNPs were detecting the same QTL. There were 44 significant SNPs identified for TC although multiple regression suggested detection of 16 separate QTLs.
These findings have important implications for macadamia breeding, and highlight the difficulties of heterozygous populations with rapid LD decay. By coupling validated marker-trait associations detected through GWAS with MAS, genetic gain could be increased by reducing the selection time for economically important nut characteristics. Genomic selection may be a more appropriate method to predict complex traits like tree size and yield.
Aims
Low-vigour scion cultivars and dwarfing rootstocks are a significant contributing factor to the success of modern temperate orchard systems. Planting density and canopy efficiency are currently ...limited in macadamia by a lack of low-vigour cultivars and rootstocks. The relationships between xylem vessel diameter, hydraulic conductivity and vigour are implicated in dwarf cultivars and low-vigour rootstocks of peach and apple. This study aims to determine relationships between vessel anatomy and tree-scale vigour in subtropical macadamia, to stimulate the development of low vigour cultivars and dwarfing rootstocks.
Methods
Stem sections from the most recent mature flush were collected from five replicates of three macadamia cultivars: ‘D4’ (high vigour), ‘B25’ (intermediate vigour) and ‘B63’ (low vigour). Sections were imaged under a fluorescence microscope in order to measure the number and size of all xylem vessels in the stem.
Results
The three cultivars showed distinct differences in height over 5 years of measurement (
P
< 0.001). Change in tree height related to the xylem vessel characteristics of mean vessel area of the ten largest vessels per section (R
2
= 0.57,
P
< 0.001), pith size (R
2
= 0.70,
P
< 0.001) and total additive vessel area (R
2
= 0.41,
P
< 0.01).
Conclusions
This study indicates that relationships between anatomical structure and vigour in macadamia are likely to be similar to those of well-studied temperate tree crops and that anatomical traits may be useful in the selection of low-vigour cultivars. Future work should investigate hydraulic relationships with vigour and rootstock-scion interactions in macadamia.
Grafting is the common propagation method for avocado and primarily benefits orchard production by reducing the time to tree productivity. It also allows use of scions and rootstocks specifically ...selected for improved productivity and commercial acceptance. Rootstocks in avocado may be propagated from mature tree cuttings ('mature'), or from seed ('juvenile'). While the use of mature scion material hastens early bearing/maturity and economic return, the molecular factors involved in the role of the scion and/or rootstock in early bearing/reduced juvenility of the grafted tree are still unknown.
Here, we utilized juvenility and flowering associated miRNAs; miR156 and miR172 and their putative target genes to screen pre-graft and post-graft material in different combinations from avocado. The abundance of mature miR156, miR172 and the miR156 target gene SPL4, showed a strong correlation to the maturity of the scion and rootstock material in avocado. Graft transmissibility of miR156 and miR172 has been explored in annual plants. Here, we show that the scion may be responsible for grafted tree maturity involving these factors, while the rootstock maturity does not significantly influence miRNA abundance in the scion. We also demonstrate that the presence of leaves on cutting rootstocks supports graft success and contributes towards intergraft signalling involving the carbohydrate-marker TPS1.
Here, we suggest that the scion largely controls the molecular 'maturity' of grafted avocado trees, however, leaves on the rootstock not only promote graft success, but can influence miRNA and mRNA abundance in the scion. This constitutes the first study on scion and rootstock contribution towards grafted tree maturity using the miR156-SPL4-miR172 regulatory module as a marker for juvenility and reproductive competence.
The Proteaceae genus Macadamia has a recent history of domestication as a commercial nut crop. We aimed to establish the first sequence-based haploid-correlated reference genetic linkage maps for ...this primarily outcrossing perennial tree crop, with marker density suitable for genome anchoring. Four first generation populations were used to maximise the segregation patterns available within full-sib, biparental and self-pollinated progeny. This allowed us to combine segregation data from overlapping subsets of >4,000 informative sequence-tagged markers to increase the effective coverage of the karyotype represented by the recombinant crossover events detected. All maps had 14 linkage groups, corresponding to the Macadamia haploid chromosome number, and enabled the anchoring and orientation of sequence scaffolds to construct a pseudo-chromosomal genome assembly for macadamia. Comparison of individual maps indicated a high level of congruence, with minor discrepancies satisfactorily resolved within the integrated maps. The combined set of maps significantly improved marker density and the proportion (70%) of the genome sequence assembly anchored. Overall, increasing our understanding of the genetic landscape and genome for this nut crop represents a substantial advance in macadamia genetics and genomics. The set of maps, large number of sequence-based markers and the reconstructed genome provide a toolkit to underpin future breeding that should help to extend the macadamia industry as well as provide resources for the long term conservation of natural populations in eastern Australia of this unique genus.
Abstract
Background
Sequencing technologies have advanced to the point where it is possible to generate high-accuracy, haplotype-resolved, chromosome-scale assemblies. Several long-read sequencing ...technologies are available, and a growing number of algorithms have been developed to assemble the reads generated by those technologies. When starting a new genome project, it is therefore challenging to select the most cost-effective sequencing technology, as well as the most appropriate software for assembly and polishing. It is thus important to benchmark different approaches applied to the same sample.
Results
Here, we report a comparison of 3 long-read sequencing technologies applied to the de novo assembly of a plant genome, Macadamia jansenii. We have generated sequencing data using Pacific Biosciences (Sequel I), Oxford Nanopore Technologies (PromethION), and BGI (single-tube Long Fragment Read) technologies for the same sample. Several assemblers were benchmarked in the assembly of Pacific Biosciences and Nanopore reads. Results obtained from combining long-read technologies or short-read and long-read technologies are also presented. The assemblies were compared for contiguity, base accuracy, and completeness, as well as sequencing costs and DNA material requirements.
Conclusions
The 3 long-read technologies produced highly contiguous and complete genome assemblies of M. jansenii. At the time of sequencing, the cost associated with each method was significantly different, but continuous improvements in technologies have resulted in greater accuracy, increased throughput, and reduced costs. We propose updating this comparison regularly with reports on significant iterations of the sequencing technologies.
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